Methods for the treatment of neuronal damage associated with ischemia, hypoxia or neurodegeneration

ABSTRACT

Intravenous administration and pharmaceutically acceptable compositions of neurotrophic factors for treating neuronal damage in the central nervous system of individuals in need of such treatment are disclosed. The neuronal damage associated with ischemia, hypoxia, or neurodegeneration may result from stroke or cardiac arrest. This invention provides for the intravenous administration of neurotrophic factors such as bFGF, aFGF, NGF, CNTF, BDNF, NT3, NT4, IGF-I and IGF-II.

This application is a 371 of PCT/US 92/09618, filed Nov. 6, 1992, whichis a continuation-in-part of U.S. application Ser. No. 07/790,734, filedNov. 8, 1991, now abandoned entitled "Intravenous Methods andPharmaceutical Composition of Neurotrophic Factors For Treatment ofNeuronal Damage Associated with Ischemia, Hypoxia or Neurodegeneration",pending.

TECHNICAL FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions useful fortreatment of neuronal damage associated with ischemia, hypoxia orneurodegeneration, and to methods for using the compositions.

BACKGROUND OF THE INVENTION

Neurotrophic factors exhibit a trophic effect on neuronal cells of thebrain. The trophic effect has been characterized as enhancing neuronalsurvival and maintenance of neuronal cell functions associated withdifferentiated neurons. In vivo studies have shown that a variety ofendogenous and exogenous neurotrophic factors exhibit a trophic effecton neuronal cells after ischemic, hypoxic or other disease-induceddamage. Examples of specific neurotrophic factors include basicfibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF),nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brainderived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4(NT4) and the insulin-like growth factors I and II (IGF-I, IGF-II).

Some neurotrophic factors, such as bFGF and CNTF, are thought to havebroad trophic effects, promoting survival or providing a maintenancefunction for many different types of neuronal cells. Other neurotrophicfactors have a narrower, more specific trophic effect and promotesurvival of fewer types of cells. For example, in the peripheral nervoussystem NGF promotes neuronal survival and axonal extension of certainspecific neuronal cell types such as sensory and sympathetic neurons(Ebendal, T. et al., Cellular and Molecular Biology of NeuronalDevelopment, Ch. 15, ed. Black, I. B., 1984). However, in the CNS, NGFalso supports the survival of cholinergic neurons in the basal forebraincomplex (Whittemore et al., Brain Res. Rev., 12:439-464, 1987). BDNF, abasic protein of molecular weight 12,300, supports some sensory neuronsthat do not respond to NGF (Barde, et al., EMBO J., 1:549-553, 1982 andHofer and Barde, Nature, 331:261-262, 1988). Neurotrophin 3 (NT3)supports survival of dorsal root ganglion neurons and proprioceptiveneurons in the trigeminal mesencephalic nucleus. CNTF, a protein ofabout molecular weight 23,000, supports ciliary ganglion neurons in theparasympathetic nervous system, sympathetic neurons, dorsal rootganglion neurons in the sensory nervous system and motor neurons in thecentral nervous system (CNS) (Kandel, E. R., et al., Principles ofNeural Science, 3rd Ed., Elsevier Science Publishing Co., Inc., NewYork, 1991).

Some neurotrophic factors constitute a family of neurotrophic factorscharacterized by about 50% amino acid homology. One such family is theBDNF/NGF family, which includes BDNF, NGF, NT3 and NT4 (Hohn, A., etal., WO 91/03569).

Fibroblast growth factors (FGFs) are members of a protein family thatinduce mitogenic, chemotactic and angiogenic activity in a variety ofcells of epithelial, mesenchymal, and neuronal origins (Zhu et al.,1990; Moscatelli, et al., U.S. Pat. No. 4,994,559). FGFs are proteins ofmolecular weight 16,000-25,000 and characterized by their strong bindingto heparin (Finklestein, S., et al., Stroke (Suppl. III)III-122-III-124, 1990; Finklestein, S. et al., Rest. Neurol. andNeurosci., 1:387-394, 1990). Both aFGF and bFGF have been reported tohave broad specificity of neurotrophic activities. Although the FGFshave similar functional activities, they are discretely differentproteins with different properties.

Because of the ability of neurotrophic factors to promote the survivalof neurons, they have been suggested to be useful for treating variousdisorders associated with neuronal cell death. Intracisternaladministration of bFGF to rats after middle cerebral artery occlusionwas reported to prevent thalamic degeneration (Yamada, K., et al., J.Cerebral Blood Flow and Met., 11:472-478, 1991). Continuousintracerebroventricular infusion of aFGF to a gerbil prevented death ofhippocampal CA1 pyramidal cells after 5 minute ischemia (Oomora, Y., etal., Soc. for Neurosci. Abstracts, 16(1):516 Abstr. No. 221.2, 1990) .

Administration of neurotrophic factors in the periphery for a site ofaction in the CNS presents several problems. For example, in vivointravenous administration of neurotrophic factors subjects them todegradative processes in the body, including proteases, which canbreakdown the protein prior to its desired action in the CNS. Manycharged molecules, including neurotrophic factors, bind to theextracellular matrix. Some neurotrophic factors affect non-neuronalcells, e.g., FGFs. Such activity may decrease the neurotrophic factor'sability to act on neuronal cells. In addition, the administration ofdrugs into the periphery for uptake into the CNS presents uniquechallenges because the blood-brain barrier prevents the entry of largemolecules into the cerebral extracellular space and cerebral spinalfluid (CSF).

CSF, found within the brain ventricles and surrounding the brain andspinal cord, is extracellular fluid that bathes neurons and glial cellsin the central nervous system. The blood-brain barrier separates theblood from direct contact with brain cells and CSF. In general, onlysmall lipophilic solutes are able to diffuse freely between the bloodvessels and the CSF. Large hydrophilic molecules, proteins and the like,generally are incapable of diffusion through the blood-brain barrier butsome are actively transported across. Generally, strongly ionized and/orlipid insoluble drugs are excluded from the brain. Non-ionized forms ofweak acids and bases are somewhat restricted, but their entry increasesin proportion to their lipid solubility. However, highly lipid solubledrugs enter the brain rapidly (Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 6th Eds, Eds. Goodman, L. S. and Gilman, A.,Macmillan Publishing Co., Inc. (1980) pp. 10, 244).

Intravenous administration of small molecules for experimental treatmentof neuronal damage associated with CNS ischemia has been done. Forexample, Kofke, W. A., et al., Stroke, 10(5):554-560 (1979) report thatthe barbiturate thiopental when administered intravenously (IV) torhesus monkeys following global brain ischemia produced bytrimethaphan-induced hypotension and a high pressure neck tourniquet,improved distribution of brain blood flow and glucose uptake.

To overcome the difficulties presented by the blood-brain barrier,molecules thought to be either too large or lipid-insoluble aregenerally administered into the CNS by direct intraventricularinjections or by way of drug impregnated implants (Otto, D., et al., J.Neurosci. Res., 22:83-91 (1989); Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 6th Ed. at 244). Another administration route isby continuous infusion through an intracerebroventricular cannula device(Williams, L. R., et al., Proc. Natl. Acad. Sci. USA, 83:9231-9235,1986). For the above reasons, neurotrophic factors have beenadministered experimentally by direct injection into the brain, byimpregnated gelfoam implants in the brain or by continuous infusion froman implanted pump into the cerebral ventricles of laboratory animalswith induced ischemic or hypoxic damage. For example, Ortiz, A., et al.,Soc. Neurosci. Abstracts, 386.18 (1990), reported that intraventricularinjection of NGF could ameliorate behavioral dysfunction associated withischemia due to middle cerebral artery and common carotid arteryocclusion.

Even though intracerebroventricular administration avoids the problemspresented by the blood-brain barrier and degradative processesassociated with an intravenous route, e.g., proteases, this route is notpreferred for routine administration to patients because it is invasive,difficult to implement and is associated with relatively high degree ofrisk compared to intravenous administration.

Chemical modification of neurotrophic factors is another approach toincreasing neurotrophic factor permeability through the blood-brainbarrier. Lewis, M. E., et al., WO 90/14838, suggest modifying IGF-I,IGF-II or NGF to increase lipophilicity, alter glycosylation or increasethe net positive charge as a means of increasing the blood-brain barrierpermeability of the neurotrophic factor.

Although some brain neurons die irrevocably soon after severe cerebralischemia, others appear to undergo a "delayed neuronal death", occurringduring the hours or days after ischemia. It is for these reasons that itis believed that there is an opportunity for therapeutic interventionfollowing stroke. The "delayed neuronal death" phenomenon appearsparticularly true for certain "selectively vulnerable" neurons followingglobal cerebral ischemia, and neurons at the borders or penumbra ofinfarcts following focal cerebral ischemia. Potential mechanisms ofdelayed neuronal death include excitatory amino acid (EAA) toxicity,free radical formation, and activation of intracellular "suicide"programs. In particular, extracellular EAA concentrations are elevatedafter transient ischemia, leading (through activation of the NMDAreceptor) to massive Ca²⁺ influx, and to subsequent initiation of lethalintracellular processes, including activation of proteases andendonucleases. Pharmacological blockade of NMDA receptor sites reducesneuronal damage in several models of focal cerebral ischemia.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition forintravenous administration comprising a neurotrophic factor in anaqueous, pharmaceutically acceptable solution.

The present invention provides a method for treating or preventingneuronal damage in the central nervous system, which method comprisesintravenous administration to a mammal in need thereof of apharmaceutical composition comprising a therapeutically effective amountof a neurotrophic factor and a pharmaceutically acceptable carrier.

This invention also provides a method for treating or preventingneuronal damage in the central nervous system comprising administeringintravenously a pharmaceutical composition comprising basic fibroblastgrowth factor (bFGF) and a pharmaceutically acceptable stabilizer thatpromotes the stability of bFGF.

More specifically, the present invention provides a method for treatingglobal or focal cerebral ischemia with bFGF administered subsequent tothe onset of ischemia. bFGF may be administered intracerebrally orintravenously up to 6 hours post ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a-b) depicts the effects of three bolus intravenousadministrations of bFGF (1.5 mg/kg/injection at 0, 24, and 48 hr. postischemia) on activity in an open field chamber and on the number ofsurviving hippocampal neurons. * indicates a significant differencebetween vehicle-treated and bFGF-treated by the unpaired t test(p<0.05). n=10 (sham), n=20 (vehicle treated), n=20 (bFGF-treated).

FIG. 2 depicts the effects of three bolus intravenous administrations ofbFGF (0.25 or 0.5 mg/kg/injection at 0, 24, and 48 hr post ischemia) onactivity in an open field chamber. n=4 (sham), n=5 (vehicle treated),n=9 (bFGF-treated, 0.25 mg/kg), and n=7 (bFGF-treated, 0.5 mg/kg).

FIG. 3(a-g) depicts the effects of three bolus intravenousadministrations of bFGF (1.5 mg/kg/injection at 2, 24, and 48 hr postischemia) on activity in an open field chamber and on the number ofsurviving hippocampal neurons. * indicates a significant differencebetween vehicle-treated and bFGF-treated by the unpaired t test (p<0.05)based on the activity parameter by the Mann Whitney test p=0.06 onneuronal counts comparing vehicle- and bFGF-treated gerbils. n=10(sham), n=17 (vehicle-treated), n=15 (bFGF-treated).

FIG. 4 depicts the effects of three bolus intravenous administrations ofbFGF (1.5 mg/kg/injection at 4 or 8 hr, 24, and 48 hr post ischemia) onactivity in an open field chamber. * indicates a significant differencebetween vehicle-treated and bFGF-treated by the unpaired t test(p<0.05). n=16 (sham), n=27 (vehicle treated), n=16 (bFGF-treated at +4hr), n=21 (bFGF-treated at +8 hr).

FIG. 5 depicts the effects of the continuous infusion of bFGF(1.5mg/kg/day for 3 days post ischemia) on activity in an open fieldchamber. n=15 (sham), n=23 (vehicle-treated) and n=20 (bFGF-treated).

FIG. 6 depicts the infarct volume of rats treated with vehicle and withbFGF as described in Example 7 below.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention relates to intravenous administration of neurotrophicfactors to treat or prevent neuronal damage resulting from ischemia,hypoxia or neurodegeneration and pharmaceutical compositions forintravenous administration comprising a neurotrophic factor. It issurprising that the neurotrophic factors reach the CNS and are stillactive in the CNS following intravenous administration. Such methods andcompositions are suitable for the treatment of stroke or cardiac arrestwhich result in ischemic or hypoxic damage or neurodegeneration andprophylactic use.

In alternate embodiments of the invention, the administration ofneurotrophic factor may be accomplished by introducing the neurotrophicfactor intracerebrally or intraventricularly. In this manner, issuesrelating to the blood/brain barrier are not relevant. However, in thepreferred embodiment the neurotrophic factors are administeredintravenously.

A neurotrophic factor is defined as any protein factor which promotesthe survival of neurons. Some neurotrophic factors are also capable ofpromoting neurite outgrowth and glial cell and blood vessel restorationor inducing cells to secrete other neurotrophic factors. Theneurotrophic factors are useful for treating neuronal damage caused bystrokes and other neurological disorders associated with generalizedcell hypoxia, ischemia or neurodegeneration. Preferred neurotrophicfactors are those to which a broad range of cell types respond. Morepreferred neurotrophic factors may be selected from the group consistingof bFGF, aFGF, CNTF, NGF, BDNF, NT3, IGF-I and IGF-II and other membersof the BDNF/NGF family. Particularly preferred neurotrophic factorsincluded bFGF, CNTF and NGF. Most preferred is bFGF.

Neurotrophic factors may be obtained from a variety of sources,preferably mammalian, most preferably human and may be isolated fromtissue or may be produced using recombinant technology. Recombinantlyproduced neurotrophic factors are preferred, and most preferred arehuman recombinantly produced neurotrophic factors. Neurotrophic factorsinclude modified fragments of the native neurotrophic factors retainingneurotrophic activity. Neurotrophic factors also include modified formsof naturally existing neurotrophic factors retaining neurotrophicactivity. Active fragments of neurotrophic factors may be identified bybioassays or receptor assays. Methods for obtaining various neurotrophicfactors are known in the art, e.g., Moscatelli, et al., U.S. Patent4,994,559 (bFGF), Collins, et al., U.S. Pat. No. 5,011,914 (CNTF), Chan,et al., EPO 370, 171 (NGF). The pharmaceutical composition may containone or more neurotrophic factors. The choice of neurotrophic factors ora mixture will be guided by the type of neuronal cells intended to betreated. The neurotrophic factors of the prevent invention may also bechemically modified according to procedures known in the art in order toenhance penetration of the blood-brain barrier.

The amount of neurotrophic factor present in the pharmaceuticalcomposition is an amount that is therapeutically effective, i.e., anamount which results in the improvement of neuronal function of neuronsdamaged by ischemia, hypoxia or neurodegeneration or prevents furtherneuronal damage. The total amount of neurotrophic factor in a dose willdepend on the specific activity of the neurotrophic factor, severity ofthe damage, responsiveness of the patient and other factors. Methods todetermine efficacy and dosage amount are known to those skilled in theart. The method of the invention provides a neurotrophic factor in adose of about 0.1 μg/kg body weight to 100 mg/kg, preferably about 1.0μg/kg to 50 mg/kg, more preferably about 10 μg/kg to 15 mg/kg and mostpreferably at least 0.2 mg/kg. The preferred total amount of bFGF whenadministration is intravenous is about 0.2 mg/kg to 10 mg/kg. A morepreferred amount of total bFGF is about 1.5 mg/kg to 4mg/kg. Thepreferred total amount of bFGF when administered intracerebrally is 0.1mg/kg to 10 μg/kg. A preferred total amount of NGF is about 1 mg/kg to50 mg/kg. A more preferred total amount of NGF is about 14 mg/kg. Apreferred total amount of CNTF is about 1 μg to 100 μg.

The timing of the initiation of the administration of the neurotrophicfactor as described herein is an important factor in effectiveness. Inthe preferred embodiment, administration is begun as soon as possiblefollowing the onset of ischemia. However, the inventors havesurprisingly found that the treatment may be effective even whentreatment is initiated as much as 6 hours post-ischemia.

In certain situations, for example in patients undergoing cardiacsurgery or who are on a heart lung machine, the neurotrophic factors ofthe present invention may be administered prophylactically. A small butsignificant percentage of those patients suffer brain ischemia duringthe procedure.

The neurotrophic factor of the present invention may be administered inthe form of a pharmaceutical composition, wherein the neurotrophicfactor is mixed with a pharmaceutically acceptable carrier. The natureof the carrier will vary depending on the nature of administration.Those skilled in the art are familiar with appropriate carriers for eachof the commonly utilized methods of administration.

The pharmaceutical composition may be an aqueous solution which maycontain pharmaceutical excipients standard for preparing intravenousinjectable or infusible compositions. The composition may contain abuffer sufficient to adjust pH of the composition to 5.0-8.0. Thephysical nature of bFGF specifically requires that the protein bestabilized prior to administration in order for the bFGF to bebiologically active as introduced into the patient. bFGF loses itsactivity rapidly in an aqueous solution, and generally must beformulated with a stabilization additive to prevent this deactivation.(See, European Patent Application 89110016.6; published as 0 345 660 ofKato et al.) Preferred stabilization additives are serum albumin,sorbitol and heparin.

A preferred ratio of neurotrophic factor to additive is from about 10:1to 1:10. A most preferred range of neurotrophic factor to additive isfrom about 5:1 to 1:5. A most preferred ratio is about 1:1.

One embodiment of this invention is a pharmaceutical composition of bFGFin an aqueous solution suitable for intravenous administration. Apreferred embodiment is a pharmaceutical composition comprising bFGF andsorbitol. The preferred composition comprises bFGF, sodium citrate,sodium chloride and sorbitol. A more preferred embodiment is apharmaceutical composition comprising bFGF with sorbitol in a ratiobetween 3:1 to 1:3 by weight. The most preferred embodiment containsbFGF and sorbitol in a 1:1 weight ratio. Another preferred compositioncomprises NGF, sodium acetate, sodium chloride and serum albumin.

One method of administering the pharmaceutical composition is byintravenous injection, but an intravenous infusion is also envisioned. Acomposition containing one or more neurotrophic factors in an aqueoussolution may be administered as a bolus injection, or a series ofinjections. The administration may be once or several times a day.

The present invention includes all forms of parenteral administration,including without limitation, intravenous, intraperitoneal,intramuscular and subcutaneous means of administration. Also includedwithin the scope of this invention are implantable polymer or membranedevices in which the neurotrophic factor is encapsulated or cellssecreting the neurotrophic factor are encapsulated. Such implantabledevices can be placed within the patient's brain or other appropriatelocations in the body.

A multitude of diseases and disorders may cause neuronal damage, inaddition to affecting other types of cells and may be treated by thecompositions and methods of this invention. The following is intended toindicate the breadth of diseases and disorders, including stroke andcardiac arrest, which cause neuronal damage and for which the presentpharmaceutical composition or the present method can be used.

Ischemia is a deficiency of blood in a tissue, due to functionalconstriction or actual obstruction of a blood vessel. Ischemia may becaused by stroke or cardiac arrest. Hypoxia, also known as anoxia, isthe reduction of oxygen supply to a tissue below physiological levels.

An important distinction should be made between global and focalcerebral ischemia. Focal ischemia, the cause of at least 500,000 casesof "stroke" seen every year in the United States, results from theblockage of a single artery in the brain, resulting in the death of allcellular elements (pannecrosis) in the territory supplied by thatartery. Global ischemia, on the other hand, results from generaldiminution of blood flow to the entire brain or forebrain, and causesthe delayed death of neurons in the "selectively vulnerable" regionsthroughout the brain. The pathology in each of these cases is quitedifferent, as are the clinical correlates. Models of focal ischemiaapply to patients with focal cerebral infarction, and models of globalischemia are analogous to cardiac arrest, and other causes of systemichypotension. It is not generally anticipated that a therapeutic would beeffective in treating both global and focal ischemic injury.

Infarction of the brain, due to focal ischemia, results when the bloodsupply to a localized area is deprived so that damage occurs to neuronaltissue. An infarct is an area of coagulation necrosis in a tissueresulting from obstruction of circulation to the area, most commonly bya thrombus or embolus. An infarct is caused by a deprivation of blood toa given area and may be either ischemic or hemorrhagic. The effect ofcerebral ischemia or hypoxia on neuronal cells depends upon itsintensity and duration and the maintenance of an effective systemiccirculation by shunting blood from other regions of the brain. Commoncauses of brain infarcts are emboli within cerebral vessels,arteriosclerotic vascular disease and the inflammatory processes, whichfrequently occur when thrombi form in the lumen of inflamed vessels.Common causes of hemorrhage into the brain are hypertension,hypertensive cerebral vascular disease, trauma rupture of aneurysms,angiomas, blood dyscrasias and bleeding from tumors. Of these,hypertension is the most common cause (Kandel, E. R., Schwartz, J. H.,Jessell, T. M., Principles of Neural Science, 3rd Ed. (1991) ElsevierScience Publishing Co., Inc., 1041-1049).

While not wishing to be bound by any theory, it is thought that cranialinjury, resulting from stroke or cardiac arrest, causing hypoxia orischemia and neurodegeneration, may make the blood-brain barrier morepermeable allowing entry into the CSF of large molecules and even highlycharged molecules which normally do not cross the barrier. It is knownthat upon injury, cells such as neutrophils and monocytes are able toenter the CSF.

Animal models have been developed which attempt to simulate neuronaldamage produced by naturally occurring ischemia, hypoxia orneurodegeneration in humans as a means to study these conditions and tostudy treatments to prevent neuronal cell damage. Some global modelsinduce neuronal damage by artificially reducing the flow of oxygen for apredetermined period of time (Pulsinelli, W. A., et al., Ann Neurol.,11:491-498, 1982). In other focal models of ischemia, a blood vessel ispermanently occluded (Finklestein, S. et al. Stroke, 21:III-122-III-124,1990; Chen, S. et al. Stroke, 17:738-743, 1986. By carefully monitoringthe conditions imposed, the location and extent of expected infarctedmaterial can be determined. Using these models, reproducible neuronaland glial cell damage can be achieved, thereby enabling the study of theefficacy of proposed pharmaceutical compositions as treatments ofneuronal damage due to ischemia.

One model of ischemic damage is the mouse middle carotid arteryocclusion model (MCA), which is a focal ischemia model (Gotti, B., etal., Brain Res., 522:290-307, 1990, which is incorporated by referenceherein in full). Another is the rat four vessel occlusion global model(Alps, B. J., et al., Neurol., 37:809-814, 1987, which is incorporatedby reference herein in full).

As described above, ischemic injury may be global or focal, that is, theareas of oxygen deprivation may include the entire brain or just localareas respectively. Focal ischemia injury may be permanent, i.e., wherea single artery is permanently occluded, or temporary where theocclusion is removed after a period of time. The effects of ischemicinjury may be different depending on the type of ischemic eventinvolved.

Methods to quantify the extent of cerebral ischemic damage are wellknown to those skilled in the art. One response to ischemic injury isthe invasion of glial cells and macrophages into the area of injury,which have significantly greater numbers of ω₃ sites than neurons. Afterischemic injury, there is a significant increase in the density of ω₃(peripheral-type benzodiazepine) binding sites (Benavides, J., et al.,Brain Res., 522:275-289 (1990)). One method to detect the presence of ω₃sites is binding assays using ³ H!-PK 11195 (New England Nuclear)(Gotti, B., et al., Brain Res., 522:290-307, 1990, which is incorporatedby reference herein in full). Another method of assessing neuronaldamage is to determine ischemic cell change (ICC) (Brown, A., et al.,Br. J. Exp. Pathol., 49:87-106, 1968, and Pulsinelli, N. A., et al.,Stroke, 10:267-272, 1979, which are incorporated by reference herein infull).

The examples found below are illustrative of different aspects of thepresent invention, each demonstrating the effectiveness of the method oftreatment described herein employing different conditions of treatmentor different animal models. Example 1 describes the intravenousadministration of bFGF and NGF in the mouse middle cerebral arteryocclusion model. In this focal model of ischemia, the middle cerebralartery is permanently occluded. The results of this experiment, whichare tabulated in Table 1, show that the administration of either bFGF orNGF beginning 15 minutes after ischemia resulted in a reduction ofischemic damage in the appropriate dosage ranges.

In Example 2, the rat four vessel occlusion global model was used, andthe neurotrophic factor examined was bFGF. This is a model of forebrainischemia where the carotid arteries are occluded temporarily. Again,bFGF administration was begun shortly following the termination of theischemic event, and some ischemia damage was prevented. The results ofthese experiments are tabulated in Tables 2 and 3.

Examples 3 and 4 describe the use of CNTF in the models described inExamples 1 and 2 respectively. Example 5 describes further experimentsutilizing the procedures described in Example 1. However, in thisexample, for one subset of mice the administration of bFGF was notinitiated until 6 hours after ischemia. Although the ischemic damage wasmore effectively prevented by a more timely administration of bFGF, theresults suggest that the initiation of administration may be at least aslong as 6 hours post ischemia. The results of these experiments arepresented in Table 4.

Example 6 employs the bicarotid occlusion model in gerbils. This is alsoa global ischemia model where both common carotid arteries are occludedtemporarily. Ischemic injury was measured by assessing the number ofsurviving pyramidal neurons in the CA1 region in the hippocampus and bymeasuring the ischemia-induced increase in hyperactivity. bFGF wasadministered intravenously either as a bolus injection or by continuousinfusion. It was shown that bFGF was effective in preventing ischemiadamage, even in cases where the onset of administration was delayed byas much as 6 hours post ischemia. The results of these experiments aredepicted in FIGS. 1-5.

In Example 7, bFGF was administered intraventricularly in a rat focalischemia model. Again, it was found that the administration of bFGFprevented some damage resulting from the ischemia. The results of theseexperiments are depicted in Table 5 and FIG. 6.

In summary, the Examples presented below show that pretreatment or posttreatment with a low dose of intravenously or intraventricularlyadministered bFGF reduces ischemic damage in a number of models.Although not limited by theory, it is likely that the effects of bFGF inreducing damage after ischemia were due directly to its potent trophiceffects on brain cells, especially neurons. High affinity receptors forbFGF are widely distributed on rat brain neurons, including those incerebral cortex. bFGF is neurotrophic for a wide variety of brainneurons in vitro (including cortical neurons), and protects culturedneurons against several neurotoxins, including EAAs, Ca²⁺ ionophore, andhypoglycemia. (Mattson, Adv. Exp. Med. Biol., 268:211-220, 1990.) Sincefailure of substrate delivery and EAA toxicity with resulting Ca²⁺ entryinto cells appear to be critical processes for neuronal death afterischemia, it is likely that the infarct-reducing effects of bFGF aredue, in part, to protection of vulnerable brain neurons, especiallythose at the borders ("penumbra") of cerebral infarcts. Theneuroprotective effects of bFGF in vitro appear to depend on new genetranscription and protein synthesis. It is thus likely that bFGF"switches on" a program of neuronal gene expression resulting in thesynthesis of "neuroprotective" proteins, and making the neurons moreresistant to ischemia. Such proteins might include heat shock proteins,Ca²⁺ buffering proteins, or Ca²⁺ /Na⁺ extrusion pumps. Moreover, theprogram of gene expression initiated by bFGF may antagonize active "celldeath" programs initiated in response to ischemia.

In addition to its trophic properties on neurons, bFGF might also reduceischemic damage through its trophic effects on brain glial cells andblood vessels. bFGF is a potent "gliotrophic" factor that promotes theproliferation of brain glial cells (including astroglia andoligodendroglia), as well as an "angiogenic" factor that promotes theproliferation of brain capillary endothelial cells and blood vessels.Thus, bFGF might protect tissue from ischemia through activation ofglial cells (in turn secreting other neurotrophic factors), or throughformation of new brain capillaries making tissue more resistant toischemia.

A third potential mechanism of bFGF neuroprotection after ischemia isalteration of systemic physiological parameters leading to decreasedinfarct size. Cuevas et al. Science, 254:1208-1210, 1991, reported thatthe intravenous administration of bFGF lowered blood pressure in maturerats and rabbits. However, decreased blood pressure, if it occurred,might be expected to increase infarct volume after focal cerebralischemia. Finally, a small but significant increase in postoperativeweight loss was found among bFGF-treated animals in Example 7 Expt. B,and a trend toward increased weight loss was found in Expt. A. bFGF andits receptor are localized in rat hypothalamus, and intraventricularadministration of bFGF has been reported to decrease food intake inmature rats. It is possible that bFGF-induced changes in food, water, orelectrolyte regulation might have influenced infarct size after focalischemia.

It has been shown that endogenous bFGF levels increase (ca. 1.5-fold) intissue surrounding focal cortical infarcts in the mature rat brain,reaching a peak at 2-3 weeks after stroke. (Finklestein, S. et al.Stroke, 21-III-122-III-124, 1990.) This "late" endogenous bFGF responsemay play an important role in wound healing, synaptic reorganization,and subsequent functional recovery after stroke. The results shown heredemonstrate that exogenous administration of bFGF appears to be usefulas a pharmacological agent to limit the extent of initial neural damageafter ischemia.

The following examples are illustrative and are not intended to limitthe scope of the invention:

EXAMPLE 1 FOCAL ISCHEMIA MODEL OF CEREBRAL ARTERY OCCLUSION IN MICE

The mouse middle cerebral artery occlusion model, MCA, is a focalischemia model that has been used to demonstrate the efficacy of bFGFand NGF in treatment of neuronal damage. A radiolabeled marker for glialcell and macrophage infiltration, 3H!-PK-11195 was used in a bindingassay to determine the extent of damage. The protocol used was adoptedfrom Gotti, B., et al., Brain Res., 522:290-307, 1990, and describes themarker, PK-11195, in greater detail.

The results show that intravenous administration of bFGF in an aqueoussolution containing the additive heparin and NGF in an aqueous solutioncontaining the additive serum albumin afford partial protection to thecerebral cortex from the effects of ischemia induced by permanentocclusion of the middle cerebral artery in the mouse.

Procedure

Adult male mice (CD₁ strain), weighing 30-40 g, were anaesthetized by anintraperitoneal (i.p.) injection of 0.1 ml 30 mg/ml pentobarbitonesodium (3 mg) or 5% Halothane in a 70%:30% Nitrous oxide:oxygen gasmixture.

The middle cerebral artery was exposed through a curved incision midwaybetween the eye and the external auditory meatus. The artery was sealedby thermocautery.

The dosing schedule was as follows. The first daily intravenous dose ofeither 1.5 μg, 15 μg, or 150 μg recombinantly produced human bFGF, 565μg recombinantly produced human NGF or placebo (all in 0.1 ml volumes)was administered about 15 minutes after ischemia. The mice were thenallowed to survive for seven days, during which single daily intravenousdoses of the same amount of either bFGF, NGF or placebo wereadministered. The intravenous solution of bFGF contained 50 mM sodiumacetate, 100 mM sodium chloride, at pH 5.0. The bFGF solutions containedan equal amount of heparin as the bFGF. The intravenous solution of NGFcontained 10 mM sodium citrate, pH 6.0, 150 mM sodium chloride in 0.1%human serum albumin. The total dosages ranges for the mice was 37.5μg/kg, 375 μg/kg and 3.75 mg/kg bFGF and 14.325 mg/kg NFG.

The animals were sacrificed 24 hours after the last dose. The infarctedarea was dissected from the infarcted hemisphere and the contralateralarea was also taken as control tissue.

Damage in the ischemic hemisphere was quantified by measuring thebinding of ³ H!-PK-11195, which provides an index of ischemic damageinsofar as an increase in binding of ³ H!-PK-11195 (assessed by B_(max))reflects neuronal damage. Compounds which prevent the increase in thenumber of binding sites are considered neuroprotective.

Results

The results of bFGF and NGF are given in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    The effect of bFGF and NGF in the MMCa-occlusion model                        Treatment                                                                           n Isc kd                                                                              MonIsc Kd                                                                           Isc Bmax                                                                            NonIsc Bmax                                                                          % inc L/R                                                                           % DAMAGE                               __________________________________________________________________________    Placebo                                                                             7 0.35 ± 0.09                                                                      0.31 ± 0.09                                                                      741 ± 166                                                                        316 ± 111                                                                          277 ± 125                                                                       100                                    1.5 μg bFGF                                                                      6 0.47 ± 0.10                                                                      0.31 ± 0.12                                                                      644 ± 171                                                                        225 ± 64                                                                           387 ± 284                                                                       99                                     15 μg bFGF                                                                       5 0.42 ± 0.04                                                                      0.22 ± 0.06                                                                      529 ± 98                                                                         218 ± 69                                                                           217 ± 79                                                                        73                                     150 μg bFGF                                                                      6 0.20 ± 0.03                                                                      0.26 ± 0.07                                                                      147 ± 23**                                                                       222 ± 57                                                                          -16 ± 17**                                                                        0                                     565 μg NGF                                                                       4 0.31 ± 0.13                                                                      0.37 ± 0.11                                                                      287 ± 144*                                                                       243 ± 128                                                                          23 ± 4*                                                                         10                                     __________________________________________________________________________     ANOVA Dunnett's test difference from placebo                                  *0.05 > p < 0.1                                                               **p < 0.01                                                               

Animals treated with placebo showing an increase in the B_(max) of ³H!-PK-11195 binding in the ischemic hemisphere resulting in an increasein the ratio of binding of the left (L, ischemic) hemisphere; right (R,non-ischemic) hemisphere. This was taken as 100% damage against whichdrug effects could be calculated. 15 μg bFGF (375 μg/kg) produced asmall but non-significant reduction in ischemic damage, however, 150μgbFGF (3.75 mg/kg) produced a total (100%) protection. 565 μg NGF (14.325mg/kg) also showed protective effects in this model.

There were no changes in the affinity of ³ H!PK-11195 for its bindingsites in the study as assessed by K_(d).

EXAMPLE 2 RAT GLOBAL ISCHEMIA MODEL OF FOUR-VESSEL OCCLUSION FOR GLOBALISCHEMIA

The rat four vessel occlusion model (4VO) was developed to quantifyneuronal damage, based on light microscopy assessment of morphologicalchanges. Comparisons of morphology are made in the presence and absenceof drug treatment. The duration of ischemic injury has been modified toparticularly affect hippocampal CA1 neurons.

Procedure

Experimental conditions were based on those described in Alps, B. J.,and Hass, W. K., Neurol., 37:809-814 (1987).

Briefly, the rats were anesthetized with Ketamine HCL (5 mg/ml, IM) and1-2% halothane. The vertebral arteries were sealed with electrocauteryfollowing the procedure outlined in Pulsinelli, W. A. and Briefly, J.B., Stroke, 10(3):267-272 (1979).

After fasting for 24 hours following sealing of the vertebral arteries,the animals were reanesthetized. During this procedure, a bitemporalelectroencephalogram (EEG) was recorded continuously. Blood pressure(BP) was recorded from a tail artery and the two common carotid arterieswere exposed for temporary occlusion.

Induction of Forebrain Ischemia

Forebrain ischemia was induced by a 10 minute occlusion of both commoncarotid arteries. During the 10 minute carotid artery occlusion, theanaesthetic concentration was varied upward or downward to hold BP atabout 50 mmHg.

Rectal temperature was measured using a digital thermometer probefollowing surgical repairs prior to returning the animals to their cagesfor recovery. Prior to this application and following the release of thecarotid artery occlusion, blood glucose was measured from an arterialsample using a Reflolux-S glucose meter (Boehringer Co London! Ltd.,UK).

Drugs, Solutions, Placebo and Treatment Regimen

Recombinantly produced human bFGF material was administered in anaqueous solution containing 100 μg/ml bFGF and 100 μg/ml heparin(Synergen, Inc., Boulder, Colo., commercially available from R&DSystems), dissolved in buffer comprising 10 mM sodium citrate (pH 6.0),150 mM sodium chloride, and 0.1% rat serum albumin. The placeboformulation given to control animals was 10 mM citrate (pH 6.0), 150 mMNaCl, 0.1% rat serum albumin.

Animals were dosed at 15 μg/kg intravenously via a tail vein within 5minutes of carotid artery reperfusion and this dose was repeated twicedaily intravenously for three days.

Termination Procedure

At 72 hours post-ischemia each animal was deeply anaesthetized withsodium pentobarbitone and thorocotomised. The cerebral circulation waswashed out with heparinized saline via cardiac puncture and the brainwas then perfused-fixed with a 10% formalin saline solution.

Neuropathological Grading System and Quantification of Damage

Neuropathological damage was assessed in terms of ischemic cell change(ICC) as described by Brown, A., and Brierley, T. B., Br. J. Exp.Pathol., 49:87-106 (1968). Specific areas examined included:

1. Layers I-VI of the striatal cortex overlying the striatum (anteriorneocortex)

2. Layers I-VI of the hippocampal cortex overlying the hippocampus(posterior neocortex)

3. The hippocampal CA1 subfield

4. The thalamus

5. The striatum

6. The cerebellar Purkinje cells and brain stem cells

Each area was graded for ICC on the basis of the following values in amanner adapted from that reported by Pulsinelli, N. A., and Brierley, J.B., Stroke, 10:267-272 (1979), which is incorporated in full. Briefly,ICC changes include:

Score 0=Normal

0-1=0-10% ICC

1-2=10-25% ICC

2-3=25-50% ICC

3-4=50-100% ICC

The scores for the hippocampal CA1 subfield, striatum and thalamus wereassessed separately for each hemisphere and the mean values derived. Thecerebellar Purkinje cells were scored for the single structure in eachanimal. Thus, for each animal a whole brain score was calculated fromthe mean of six regional values and then averaged to give a group meanscore both including and omitting the CA1 component. An absolute cellcount was also made for the CA1 subfield.

General Hemodynamic Data for Blood Pressure, Body Temperature and BloodGlucose

Blood pressure was measured but did not change. Data for the bloodpressure in the rat is presented in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    BLOOD PRESSURE (STYSTOLIC/DIASTOLIC BP) (mmHg)                                IN RATS SUBJECTED TO 10 MIN. FOREBRAIN ISCHEMIA                                                       Isoelectric                                                Starting                                                                            Pre-occlusion                                                                        Peak Pressor                                                                        EEG   Return to EEG                                                                        Final                                    Group                                                                              BP    BP     BP    BP    BP     BP                                       __________________________________________________________________________    Drug-                                                                               *85.8 ± 5.4                                                                     75.8 ± 6.9                                                                        109.0 ± 8.1                                                                      75.8 ± 14.1                                                                      118.3 ± 5.9                                                                       118.3 ± 5.9                           treated                                                                            **80.8 ± 5.4                                                                     70.8 ± 6.9                                                                        104.5 ± 8.5                                                                      70.8 ± 14.1                                                                      113.3 ± 5.9                                                                       113.3 ± 5.9                           bFGF                                                                          (n = 6)                                                                       Vehicle                                                                             *85.4 ± 3.2                                                                     77.0 ± 7.2                                                                        106.0 ± 6.8                                                                      97.0 ± 13.2                                                                      129.0 ± 4.3                                                                       133.0 ± 2.5                           Controls                                                                           **82.4 ± 4.0                                                                     72.0 ± 7.2                                                                        101.0 ± 6.8                                                                      92.0 ± 13.2                                                                      124.0 ± 4.3                                                                       128.0 ± 2.5                           (n = 5)                                                                       __________________________________________________________________________     *systolic                                                                     **diastolic                                                              

Rectal body temperatures measured at the end of ischemia wereacceptable, with no evidence of hypothermia nor hyperthermia. The meandrug-treated animal temperature was 37.7°±0.12° C. and for vehiclecontrols was 37.9°±0.18° C.

Blood glucose levels were unaffected by the procedure in either group,where the drug-treated animals showed a pre-ischemia value of 7.5±0.2 mMand post-ischemia value of 7.0±0.3 mM, and where these respective valuesfor vehicle controls were 7.6±0.5 mM and 7.5±0.3 mM.

Neuropathological Findings and ICC Scores

Data for the effects of bFGF against ICC compared to untreated controlsis presented in Table 3. Data for normal brains is also included inTable 3 to indicate the background level for artifact staining.

                                      TABLE 3                                     __________________________________________________________________________    NEUROPATHOLOGICAL SCORES (0-4) FOR ISCHEMICA CELL CHANGE IN RATS              SUBJECTED TO FOUR VESSEL OCCLUSION AND TREATED WITH bFGF VERSUS DRUG          VEHICLE ALONE                                                                      Hippocampal                                                                         Posterior                                                                           Anterior          Cerebellar                                                                           Mean Brain Score                    Group                                                                              CA.sub.1 Cells                                                                      Neocortex                                                                           Neocortex                                                                           Thalamus                                                                            Striatum                                                                            Purkinje Cells                                                                       +CA.sub.1                                                                            -CA.sub.1                    __________________________________________________________________________    Control                                                                            4.00 ± 0.00                                                                      1.40 ± 0.20                                                                      1.15 ± 0.20                                                                      1.60 ± 0.20                                                                      1.35 ± 0.20                                                                      1.70 ± 0.30                                                                       1.85 ± 0.10                                                                       1.44 ± 0.11               n = 5                                                                         bFGF 2.58 ± 0.63                                                                      0.75 ± 0.26                                                                      0.92 ± 0.15                                                                      0.71 ± 0.48                                                                      0.50 ± 0.34                                                                      1.17 ± 0.53                                                                       1.10* ± 0.31                                                                      0.81 ± 0.33               (n = 6)                                                                       % Control                                                                          64.5% 53.6% 80.0% 44.4% 37.0% 68.8%  58.1 ± 6.6%                                                                       56.8 ± 7.5%               Score                                                                         Normal                                                                             0.33 ± 0.11                                                                      0.31 ± 0.06                                                                      0.36 ± 0.12                                                                      0.79 ± 0.19                                                                      0.79 ± 0.19                                                                      0.50 ± 0.18                                                                       0.52 ± 0.09                                                                       0.55 ± 0.09               (n = 6)                                                                       __________________________________________________________________________     *Statistical Significance P <0.05 (ttest)                                

The hippocampal CA1 neurons were partially protected in animals treatedwith bFGF compared to surviving controls, which all showed maximaldamage on subjective scoring, thus there was no standard error and theregion was re-evaluated by an absolute cell count. The percentage ofabnormal neurons in the vehicle control group was 97.7±0.8% ICC (n=5)and the bFGF-treated group was 56.6±16.8% ICC (n=6) (p<0.5 t-test). Allother brain areas examined showed partial protection for bFGF. Overall,the drug-treated group showed 58.1±6.6% of the level of the controlgroup mean brain score for damage with the CA1 component value includedand 56.8±7.9% in the absence of the CA1 value. Protection for thethalamus and striatum compared favorably against ICC scores for normalbrains. These data show that bFGF administered intravenously prevents orreduces the incidence of delayed neuronal degeneration which occurs overthree days following an ischemic insult in the rat and that the compoundmay be effective in several brain areas.

Morphological assessment of brain slices demonstrated that intravenousadministration of neurotrophic factors partially protected hippocampalCA1 neurons in the rat 4VO model, as were the regions including theanterior and posterior neocortex, thalamus, striatum and cerebellarPurkinje cells, against ischemia-induced damage (Table 3).

EXAMPLE 3 ADMINISTRATION OF CNTF IN THE MOUSE FOCAL ISCHEMIA MCA MODEL

The effects of CNTF are studied in the mouse MCA occlusion modelessentially following the procedure set forth in Example 1. CNTF isstudied in these animal models using pharmaceutically acceptablecompositions in the following daily amounts, 1 μg, 10 μg or 100 μg. Theamount of CNTF may be adjusted to provide equivalent dosages accordingto the weight of the individual being treated.

EXAMPLE 4 ADMINISTRATION OF CNTF IN THE RAT GLOBAL ISCHEMIA 4VO MODEL

The effects of CNTF are studied in the rat 4VO model essentiallyfollowing the procedure set forth in Example 2. CNTF is studied in theseanimal models using pharmaceutically acceptable compositions in thefollowing daily amounts, 1 μg, 10 μg, or 100 μg. The amount of CNTF maybe adjusted to provide equivalent dosages according to the weight of theindividual being treated.

EXAMPLE 5 DELAYED bFGF ADMINISTRATION IN MCA MODEL OF FOCAL ISCHEMIA INMICE

The middle carotid artery occlusion model (MCA) of focal ischemia asdescribed in Example 1 was performed to determine if the delayedadministration of bFGF could reduce ischemic damage. The results of thisseries of experiments are summarized in Table 4. Results are given formice that were given intravenously a placebo after the ischemic event,mice given intravenously 100μg of bFGF 15 minutes, 24 h and 48 hpost-ischemia, and mice given intravenously 100 μg of bFGF 6 h, 24 h and48 h post-ischemia.

Consistent with the results shown in Example 1, mice given bFGF shortlyafter the ischemia event were strongly protected from neuronal damage.Those mice given their first dose of bFGF 6 hours post-ischemia werealso provided some protection from neuronal damage.

The measure of % damage as given in Table 4, is an index of protectionwhich takes into consideration both the absolute values and the %increase L/R; any changes in right hemisphere are corrected for

% damage =(left_(t) -right_(t) /left_(c) -right_(c)) % where c is thecontrol (placebo) group and t is the treatment group.

                  TABLE 4                                                         ______________________________________                                                 Bmax         % increase                                              n          left      right    L/R     % damage                                ______________________________________                                        Placebo  9     577 ± 128                                                                            128 ± 34                                                                          699 ± 365                                                                          100                                   100 μg bFGF                                                                        10     220 ± 60                                                                             107 ± 19                                                                          99 ± 38                                                                            25                                    (15 min,                                                                      24 h, 48 h)                                                                   100 μg bFGF                                                                        10     495 ± 103                                                                            166 ± 31                                                                          294 ± 120                                                                          73                                    (6 h, 24 h,                                                                   48 h)                                                                         ______________________________________                                    

EXAMPLE 6 EFFECTS OF INTRAVENOUSLY ADMINISTERED bFGF ON GLOBAL ISCHEMICINJURY IN THE GERBIL

The mongolian gerbil is a useful model for studying ischemic injury.Ischemic injury can be induced by occluding blood flow through bothcommon carotid arteries (bicarotid occlusion, BCO) since there is anincomplete circle of Willis connecting the carotid and vertebrobasilararteries. Neurons in the CA1 region in the hippocampus of gerbils areselectively vulnerable to ischemia and undergo a slow process of delayedneuronal degeneration. Depending upon the duration of bicarotidocclusion, other forebrain structures such as the striatum, thalamus,and neocortical layers 3 and 5 may also be damaged. In this example, theeffect of intravenously administered bFGF was tested on two ischemicinjury parameters; 1) an ischemia-induced decrease in the number ofsurviving pyramidal neurons in the CA1 region in the hippocampus and 2)an ischemia-induced increase in hyperactivity in an open-field chamber.

In this example, the intravenous injection of bFGF is judged to beneuroprotective if treated gerbils meet the following criteria; 1) thereis a bFGF-induced increase in the number of neurons in histologicalsections of hippocampus taken at seven days post ischemia; and 2) thereis a bFGF-induced decrease in an abnormal ischemia-induced behavioralparameter, hyperactivity in an open-field chamber. Lesioning of thehippocampus causes impaired spatial learning in monkeys and rats. In thegerbil, transient forebrain ischemia causes an impairment in spatialmapping such that gerbils with ischemic injury display persistentexploration of a test environment (Chandler, M. J. Pharm. Meth.14:137-146, 1985 and Wang, D. Brain Research, 533:78-82, 1990). Theincrease in exploratory behavior in an open-field chamber reflects theimpaired ability of an ischemia-injured gerbil to make a mental map of anovel environment. Thus, the measurement of open-field activity afterischemia allows the assessment of cognitive functions in the hippocampusand supports the assessment of histological preservation of hippocampalneurons.

Ischemic Injury

On the day before the global ischemia, gerbils were anesthetized withisoflurane/oxygen and a ventral midline incision was made in thecervical region. Both carotid arteries were identified and looselylooped with silk suture. The external jugular vein was cannulatedunilaterally with polyethylene tubing. The tubing was exteriorized atthe nape of the neck to facilitate the intravenous injection of bFGF orvehicle. On the following day, the instrumented gerbils wereanesthetized with isoflurane/oxygen and the cervical incision wasreopened. The right and left common carotid arteries were identified bythe presence of the looped silk suture and were occluded for 2 min 25sec with microaneurysm clips. Throughout the ischemia, body temperaturewas maintained at 37.5° C. After blood flow was reestablished throughthe carotid arteries and the cervical incision was closed, the gerbilswere placed for two hours in an incubator that was maintained at 37.5°C. Sham-treated gerbils underwent cannulation of the jugular vein,looping of the carotid arteries, and exteriorization of the carotidarteries without occlusion of blood flow.

Treatment with bFGF

bFGF was administered intravenously as either a bolus injection or bycontinuous infusion. Human recombinant bFGF, from Synergen, Inc., wasused in a saline formulation for bolus injection and with sorbitol as astabilizing agent for infusion. The bolus injections were given toisoflurane-anesthetized gerbils into the jugular vein through theexteriorized polyethylene tubing. The continuous infusion of bFGF intothe jugular vein was accomplished by connecting the indwellingpolyethylene tubing to a mini-osmotic pump. The gerbils receiving bolusinjections were treated three times at approximately 24 hr intervals.The first treatment was given at either 0, +2 hr, +4 hr or +8 hrrelative to the bicarotid occlusion. The second and third treatmentswere given at 24 and 48 hr post bicarotid occlusion. The continuousinfusion of drug was at a delivery rate of one microliter per hour froman Alzet 2001 mini-osmotic pump such that a total dose of 1.5 mg bFGF/kgper day was delivered for three days.

Assessment of Ischemic Injury

The activity of the gerbils in an open-field chamber was measured at 96hr post bicarotid occlusion. The gerbil was placed in the griddedchamber and its movements were recorded for 15 min on a videocamera.Activity was expressed as the number of squares crossed during the finalten min period of observation in the chamber. On day seven, hippocampaltissue was obtained for histological assessment after perfusion fixationof the brain by the intracardiac injection of a buffered formalinsolution. Paraffin-embedded sections were stained with cresyl violet. Ablinded observer counted the number of histologically preserved neuronsin the CA1 region of the right and left hippocampi. The reported valuesare combined counts from a one mm section from each hemisphere. Invehicle-treated ischemic gerbils, cell loss resulting from necrosis andphagocytosis of the neurons was almost complete from thehypoxia-vulnerable region of the hippocampus. The total number ofneurons remaining in vehicle- and bFGF-treated gerbils was compared.

Results of Bolus Intravenous Administration of bFGF.

The effects of the bolus intravenous administration of bFGF at doses of0.25, 0.5, and 1.5 mg/kg was tested. The bFGF was administered at 0, 24,and 48 hr post ischemic injury. FIG. 1 shows the effect of the highestdose on ischemia-induced hyperactivity and neuronal loss. At 1.5 mg/kg,bFGF significantly decreased the hyperactivity and increased the numberof surviving neurons. At doses of 0.25 and 0.5 mg/kg, treatment withbFGF was not effective in reducing ischemic injury (FIG. 2). Theprotective effects of bFGF at 1.5 mg/kg when the first treatment wasdelayed until 2, 4, or 8 hr post ischemia and the second and thirdtreatments with bFGF are given at 24 and 48 hr post ischemia was alsotested. At the 2 hr timepoint, bFGF was protective against ischemicinjury based on a significant reduction in hyperactivity and asignificant salvage of hippocampal neurons (FIG. 3). At the 4 and 8 hrtimepoints, bFGF was protective against ischemic injury based on asignificant reduction in hyperactivity (FIG. 4).

Results of Continuous Intravenous Infusion of bFGF

The protective effects of bFGF administered by continuous intravenousinfusion with the onset of delivery at time zero relative to theischemic injury and the rate of delivery at 1.5 mg/kg during each 24 hrperiod was also performed. Under these conditions, bFGF was notprotective against ischemic injury when continuously infused based onthe hyperactivity parameter (FIG. 5).

EXAMPLE 7 bFGF ADMINISTERED INTRAVENTRICULARLY IN RAT FOCAL ISCHEMIAMODEL

Mature male Long-Evans rats (250-350 g) were anesthetized with sodiumpentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic head holder(David Kopf Instruments, Tujuna, Calif.). The dorsal surface of theskull was exposed by midline incision, and a small burr hole (2 mmdiameter) was drilled over the right lateral ventricle, 1.6 mm lateraland 0.9 mm posterior to bregma. A stainless steel cannula (I.D. 0.020",O.D. 0.028", 2 cm long) was then inserted stereotaxically into theventricle to a depth of 4.4 mm beneath the surface of the skull. Thetubing was bent at a 90° angle 1-1.5 cm from its tip and connected topolyethylene tubing (I.D. 0.76 mm, O.D. 1.22 mm, 10 cm long) that wasconnected (by glue) to a mini-osmotic pump (Alzet 1007D, 100 μl fillvolume, pump rate =0.5 μl/hr; Alza Corp., Palo Alto, Calif.) implantedsubcutaneously in the back. The cannuia was fixed to the skull byorthodontic resin (L.D. Culk Co., Milford, Del.) bonded to two smallmachine screws (1/8" stainless steel slotted) inserted in the skull. Thepump, tubing, and cannula were primed before insertion with infusatesolutions; a 3-0 nylon suture was inserted into the cannula duringimplantation to prevent obstruction by brain tissue. The wound wasclosed with 3-0 silk suture and cefazolin (10 mg, i.m.) wasadministered). After surgery animals were kept in individual cages andfed soft food.

For Experiment A (Expt. A), pumps were filled with vehicle alone(containing 127 mM NaCl, 2.6 mM KCl, 1.2 mM CaCl₂, 0.9 mM MgCl₂, 4.14 mMHEPES, 3 mM glycerin, 0.001% bovine serum albumin BSA!, and 0.01% fastgreen), or vehicle plus bFGF (100 μgm/ml). For Experiment B (Expt. B),pumps were filled with vehicle alone (containing 143 mM NaCl, 2.6 mMKCl, 1.3 mM CaCl₂, 0.9 mM MgCl₂, 4.6 mM HEPES, 1.0 mM sodium citrate,0.01% human serum albumin HSA!, and 0.01% fast green), or vehicle plusbFGF (100 μgm/ml) plus heparin (100 μgm/ml), added to stabilize bFGF. Atthe concentration and pump rate used, bFGF was delivered at 50 ng/hr, or1.2 μgm/day. For each experiment, the vehicle represented artificialCSF, plus albumin as protein carrier, plus fast green as a dye tovisualize cannula placement, plus trace concentrations of bufferspresent in bFGF stock solutions. The differences in vehicles for Expts.A and B reflect differences in the bFGF stock solutions used in theseexperiments. Recombinant human bFGF was obtained from Synergen, Inc.(Boulder, Colo.). Each batch of bFGF used was tested for biologicalactivity by standard mitogenic assay on Balb c/3T3 cells (5-10 activityunits/ng). The heparin preparation used (porcine intestinal heparin;Biosynth International, Skokie, Ill.) had no biological activity in thisassay.

Three days after cannula implantation, animals were reanesthetized with2% halothane and given atropine (0.15 mg/kg, i.p.). Animals were thenintubated and connected to a ventilator (SAR-830; CWE Inc., Ardmore,Pa.) delivering 1% halothane/70% nitrous oxide in oxygen. The rightfemoral artery and vein were cannulated for monitoring of mean arterialblood pressure (MABP; Gould RS3200 Blood Pressure Monitor, Gould Inc.,Valley View, Ohio), drug delivery, and blood sampling. Animals were thenparalyzed with pancuronium bromide (0.5 mg/kg, i.v.) Arterial bloodgasses (Corning 178 Blood Gas Analyzer, Ciba Corning Diagnostic Corp.,Medford, Mass.), blood glucose (Accu-Check Blood Glucose Analyzer,Boehringer Mannheim, Indianapolis, Ind.), and hematocrit were measuredat least twice during surgery and the immediate post-operative period.The stroke volume and rate of the ventilator was adjusted to maintainPaO₂ between 100-200 mmHg and PaCO₂ between 30-40 mm Hg. Core bodytemperature was monitored by rectal thermocouple (Model 73 ATA, YellowSprings Instrument Co., Yellow Springs, Ohio) and maintained between36°-37.5° C. with a homeothermic blanket control unit (HarvardBioscience, South Natick, Mass.).

Focal ischemic infarcts were made in the right lateral cerebral cortexin the territory of the middle cerebral artery (MCA) by the method ofChen, et al. Stroke, 17:738-743, 1986. Both common carotid arteries wereexposed by midline ventral incision. The animal was then placed in astereotaxic head holder, and a 1 cm skin incision was made at themidpoint between the right lateral canthus and the anterior pinna. Thetemporal muscle was retracted, and a small (3 mm diameter) craniectomywas made at the junction of the zygoma and squamosal bone using a dentaldrill cooled with saline. Using a dissecting microscope, the dura wasopened with fine forceps, and the right MCA was ligated with two 10-0monofilament nylon ties just above the rhinal fissure and transectedbetween the ties. Both common carotid arteries were then occluded bymicroaneurysm clips for 45 min. After removal of the clips, return offlow was visualized in the arteries. Anesthesia was maintained for 15min., and animals were returned to individual cages and fed soft foodafter surgery.

Twenty four hours after cerebral infarction, animals were again weighed,and then sacrificed by rapid decapitation. In some animals, rectaltemperatures was also measured before sacrifice. Brains were removed,inspected visually for anatomy of the MCA as well as for signs ofhemorrhage or infection, immersed in cold saline for 10 min., andsectioned into seven standard coronal slices (each 2 mm thick) using arodent brain matrix slicer (Activational Systems, Warren, Mich.). Brainswere also examined visually for the presence of dye (fast green) in thecerebral ventricles, basal cisterns, and over the convexities. Sliceswere placed in the vital dye, 2,3,5-triphenyl tetrazolium chloride (TTC,2%; Chemical Dynamics Co., N.J.) at 37° C. in the dark for 30 min.,followed by 10% formalin at room temperature overnight. The outline ofright and left cerebral hemispheres as well as that of infarcted tissue,clearly visualizable by lack of TCC staining, was outlined on theposterior surface of each slice using an image analyzer (Olympus SZHmicroscope connected to an MTI videocamera and Sony video monitor;Bioquant IV Image Analysis System run on an EVEREX computer). Both thesurgeon and image analyzer operator were blinded to the treatment giveneach animal.

To test the specific hypothesis that bFGF treatment reduces infarctvolume, volume of infarcts among bFGF-vs. vehicle-treated animals werecompared by unpaired, two-tailed t-tests for each experiment, and bytwo-way analysis of variance (ANOVA; Expt. X Treatment) for combineddata. Other anatomical and physiological measurements were comparedamong bFGF-vs. vehicle-treated animals by unpaired, two-tailed t-testsfor each experiment, using the Bonferroni correction for multiplepairwise comparisons.

RESULTS

Of 63 animals prepared initially for Expts. A and B, five (8%) diedbefore sacrifice; three (5%) were excluded because of intra-operativehypoxia, hypercapnia, or hypotension; three (5%) were excluded due tolack of dye in ventricles on sacrifice; and four (6%.) were excludedbecause of anatomical anomalies of the middle cerebral artery ("doubleMCA") discovered on sacrifice. Data on the remaining 48 animals (76% oftotal prepared) were taken for analysis. Excluded animals weredistributed equally among vehicle- and bFGF-treated animals, and amongExpts. A and B.

The model of focal cerebral ischemia used resulted inclearly-visualizable, well-demarcated infarcts in the right lateralcerebral cortex in the territory of the MCA. All brains taken foranalysis showed the presence of dye (fast green) in the cerebralventricles, as well as faint dye in the basal cisterns and over theconvexities. No gross infections or hemorrhages into infarcts was foundat sacrifice. Overall infarct volume was 110±6 mm³ (mean ±SEM; N=48).

In Expt. A, infarct volume in animals receiving vehicle alone (N=12) orvehicle plus bFGF (1.2 μgm/day; N=12) was compared. This experimentshowed a 27% reduction in infarct volume among bFGF-vs. vehicle-treatedanimals (98±14 vs. 135±11 mm³ mean ±SEM, respectively; t=2.07, df=22,p=0.05; FIG. 6). In Expt. B, this same dose of bFGF was co-administeredwith an equivalent dose of heparin, added to stabilize the bFGF. (Theheparin preparation used had no FGF-like activity as assessed bystandard Balb c/3T3 mitogenic assay, and, at the low dose administered,we found no evidence of the anticoagulant effects of heparin in terms ofmacro- or micro-hemorrhage into infarcts.) Expt. B showed a 21%reduction in infarct size among bFGF/heparin (N=13) vs. vehicle-treated(N=11) animals (92±7 vs 117±10 mm³, mean±SEM, respectively; t=2.11,df=22, p=0.05; FIG. 1). Differences in infarct volumes for Expts. A andB were not accounted for by differences in right hemisphere or totalbrain volume among animals (Table 5).

A two-way ANOVA showed no effect of experiment on lesion volume (F1/44!=1.22,p-n.s,), or interaction between experiment and treatment (F1/44!=0.29,p-n.s.), but did confirm an overall treatment effect in favorof bFGF (25% reduction in infarct volume; 126±8 vs. 95±7 mm³ forvehicle- N=23! vs. bFGF-treated animals N=25!, respectively; F1/44!=8.35, p=0.006).

No differences in intraoperative MABP, arterial PaO₂, PaCO₂ or pH, bloodglucose, hematocrit, or core temperature were found among vehicle- vs.bFGF-treated animals for Expt. A or B (Table 5). Moreover, we found nodifferences in postoperative core temperature in a random subset ofvehicle- vs. bFGF-treated animals in Expt. B (37.8±0.1 N=4! vs.37.4±0.1° C. N=5!, respectively; t=1.81, df=7, p-n.s.). A small (14%)but significant increase in postoperative weight loss was found amongbFGF-treated animals in Expt. B, and there was a trend toward increasedweight loss in Expt. B (Table 5). However, the magnitude ofpostoperative weight loss was not correlated with infarct volume eitherfor Expt. B alone (r=0.314, p-n.s.; N=24), or for the combined results(r=0.035, p-n.s.; N=48).

                  TABLE 5                                                         ______________________________________                                        EXPT. A             EXPT. B                                                   Vehicle       bFGF      Vehicle   bFGF                                        (N = 12)      (N = 12)  (N = 11)  (N = 13)                                    ______________________________________                                        Preoperative                                                                  Weight (gm)                                                                           288 ±                                                                              8     292 ±                                                                            6   284 ±                                                                            6   280 ±                                                                            6                             Intra-                                                                        operative                                                                     MABP    106 ±                                                                              4     113 ±                                                                            4   110 ±                                                                            4   123 ±                                                                            2                             (mm Hg)                                                                       PaO2 (torr)                                                                           145 ±                                                                              6     147 ±                                                                            6   172 ±                                                                            7   165 ±                                                                            7                             PaCO2 (torr)                                                                          34 ± 1     35 ±                                                                             1   35 ±                                                                             1   33 ±                                                                             1                             pH      7.39 ±                                                                             .01   7.42 ±                                                                           .01 7.43 ±                                                                           .01 7.44 ±                                                                           .01                           Glucose 126 ±                                                                              2     128 ±                                                                            4   137 ±                                                                            7   132 ±                                                                            5                             (gm/dl)                                                                       Hematocrit                                                                            40 ± 1     40 ±                                                                             1   39 ±                                                                             1   42 ±                                                                             1                             Temperature                                                                           37.0 ±                                                                             0.1   37.0 ±                                                                           0.1 36.8 ±                                                                           0.1 36.7 ±                                                                           0.1                           (°C.)                                                                  Post-                                                                         operative                                                                     Weight  -30 ±                                                                              8     -51 ±                                                                            5   -10 ±                                                                            6   -40 ±                                                                            5**                           Change (gm)                                                                   R Hem.  696 ±                                                                              19    689 ±                                                                            16  711 ±                                                                            8   716 ±                                                                            22                            Vol. (mm3)                                                                    L Hem.  652 ±                                                                              18    651 ±                                                                            19  677 ±                                                                            13  686 ±                                                                            18                            Vol. (mm3)                                                                    Total Vol.                                                                            1348 ±                                                                             36    1340 ±                                                                           34  1389 ±                                                                           19  1402 ±                                                                           40                            (mm3)                                                                         ______________________________________                                    

We claim:
 1. A method of preventing the occurrence, or limiting thesize, of a region of substantially complete cell death in the brain dueto focal ischemic injury, comprising: selecting a mammal that hassuffered, or who is at risk of suffering, such a region of substantiallycomplete cell death, and administering bFGF to said mammal intravenouslyin sufficient dosage to take effect across the blood-brain barrier toeffectively prevent the occurrence or limit the size of said region. 2.The method of claim 1, further comprising selecting a mammal that hassuffered, or who is at risk of suffering, from constriction orobstruction of a single arterial blood vessel in the brain.
 3. Themethod of claim 1, further comprising selecting a mammal that hassuffered, or who is at risk of suffering, from hemorrhage into thebrain.
 4. The method of claim 3, wherein said hemorrhage is caused byhypertension, hypertensive cerebral vascular disease, trauma, rupture ofaneurysms, angiomas, blood dyscrasias, or bleeding from tumors.